1 / 55

Unit 6: Regulatory Circuits

Unit 6: Regulatory Circuits. Gene Expression: From DNA to Protein. Replication (+repair). DNA. Transcription. hnRNA. Splicing. mRNA. Translation. Polypeptide. M. L. I. V. G. Folding, modification. Folded, mature Protein. Gene Expression: From DNA to Protein.

pepper
Download Presentation

Unit 6: Regulatory Circuits

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Unit 6: Regulatory Circuits

  2. Gene Expression:From DNA to Protein Replication(+repair) DNA Transcription hnRNA Splicing mRNA Translation Polypeptide M L I V G Folding, modification Folded, mature Protein

  3. Gene Expression: From DNA to Protein • We will shift our level of abstraction • Before, we treated each DNA, RNA or amino acid monomer as our essential process • Now, we will treat functional units as essential processes • Genes • RNA molecules • Proteins

  4. mRNA degradation control mRNA translation control Multi-Step Regulation of Gene Expression nucleus cytosol Degraded mRNA Primary RNA transcript DNA mRNA mRNA Transcriptioncontrol RNA processingcontrol RNA transportcontrol Protein protein activity control Active Protein Protein degradation control Degraded Proteinn

  5. Two Views of Regulation • The biochemical view: What are the specific biochemical events that regulate gene expression? E.g. how is transcription regulated? • The logical view: What is the functional outcome of a regulatory event: up-regulation (activation) or down-regulation (inhibition)?

  6. Basal Transcription TFIID binds specifically to TATA sequence TFIIB enters the complex PolII-TFIIF enter TFIIE and H enter TFIIH phosphorylates PolII;Pol II is released from the TF complex and can start transcription

  7. Regulation of Transcription: Biochemical View Specific transcription factors

  8. Transcription Factors • Most transcription factors are DNA-binding proteins • They recognize and bind regulatoryDNA motifs: relatively specific short sequences of DNA • These regulatory sequences may be in the vicinity of the promoter (RNAPII binding site) or at a distance (enhancers)

  9. Transcription Factors • Transcription factors modulate basal transcription levels, either activating or repressing it • In many cases this is mediated by facilitating (positive control) or hindering (negative control) the assembly of the general transcription complex • The ability of the regulatory protein to bind to DNA is often regulated as well

  10. The Principles: Prokaryotic Regulation of Transcription

  11. Example: The Tryptophan Repressor The Trp operon: Several enzymes for tryptophan biosynthesis under one promoter The Trppromoter: A repressor-trp complex binds and prevents Pol-II binding

  12. Activators and Repressors A repressor prevents transcription when bound to DNA. An activator enhances transcription when bound to DNA. A small molecule may bind and change the conformation of the TF. In some cases this will promote binding. In others it will inhibit it.

  13. Activator and Repressor ON The function of the “lambda repressor” protein depends on its precise placement on its binding site OFF

  14. Activator and Repressor ON The function of the “lambda repressor” protein depends on its precise placement on its binding site OFF

  15. Multiple Controls:The lac Operon “logic” Lactose binds to the lac repressor and removes it from the promoterGlucose decreases the concentration of cAMP, which releases CAP, causing it to unbind from the promoter. Only when there is lactose and there is no glucose will the CAP be bound and the repressor not, and the operon will be ON

  16. Regulation At a Distance: Enhances NtrC is a bacterial regulatory protein that activates transcription by interacting with the PolII complex. NtrC is bound to a distant enhancer. The DNA loops out to allow NtrC at the enhancer to interact with the PolII complex at the promoter The DNA serves as a tether that facilitates interaction between different bound proteins

  17. Eukaryotic Transcription • Most prokaryotic genes are controlled by only one or two gene regulatory proteins • The regulation of higher eukaryotic genes is much more complex, where tens of transcriptional activators and repressors may bind to a large regulatory region • For example, the control region of the eve gene of the fruit fly (Drosophila) is 20,000 nt long, and has binding sites for over 20 proteins

  18. Gene Control Region regulatory sequences promoter

  19. Eukaryotic Activators Many activators accelerate the assembly of general transcription factors. Most have a modular design. In the simplest cases, one part (binding domain) of the protein recognizes the regulatory site and binds to the DNA, while another activation domain, contacts the transcription mahcinery and accelarates initiation.

  20. Eukaryotic Repressors • Repressors may work in several ways: • Compete with an activator on overlapping sites. • Bind the activation site of an activator, preventing its interaction with the transcription machinery • Directly interact with the transcription machinery, blocking assembly

  21. Combinatorial Regulation • This combination of multiple regulatory sites (and proteins) for a single gene allows fine-tuning of transcription and its levels • The action of many transcription factors depends on the formation of the correct complex at the regulatory site. • For example, some factors do not bind to DNA directly but are recruited by binding to other DNA binding proteins. • The same protein may serve as an activator or repressor, depending on the context in which it is present

  22. Context-Dependent Function Some activator proteins do not directly interact with the PolII complex, but rather mediate the binding of other proteins (one contributes a DNA binding domain, and another an activation domain) The green protein may be part of an activation or repression complex, depending on the context in which it binds to the DNA: What other proteins are present? What other binding sequences are in the promoter?

  23. Combinatorial Regulation Multiple sets of proteins, organized in several “modules”, can work together to influence a promoter. It is not yet understood in detail how the integration of multiple inputs is achieved.

  24. Combinatorial Regulation • The activity of a gene regulatory protein can itself be regulated, e.g. • Transcriptionally – control over the quantity of proteins • Post-translationally – control over the quantity of active (binding-enabled) protein in the nucleus • This leads to the formation of complex regulatory circuits

  25. Regulating regulatory proteins Change in conformation by ligand binding. Only bound protein can bind DNA Only dimer complex of two proteins can bind DNA In order to bind DNA, the protein must first be translocated to the nucleus How much protein is created?Transcription, splicing, degradation, translation Change in conformation by protein phosphorylation. Only phospho-protein can bind DNA Binding site is revealed only after removal of an inhibitor

  26. The lac Operon “logic” By using two regulatory proteins (CAP and Lac repressor), and controling their binding by two different sugars, transcription is accurately controlled: Only when glucose is absent and lactose is present the operon is switched ON Activates lactose metabolism only when there is lactose to metabolize and there is no glucose to metabolize

  27. The Lysis/lysogeny logic(Shapiro and McAdams, 1995; Arkin et al 1998)

  28. The Cell Cycle “logic” Kohn, 1999

  29. The Cell Cycle Logic (cont) Kohn, 1999

  30. Modeling Approaches • Logical – treat as Boolean (AND, OR, NOT etc) system • Biochemical – model biochemical events as accurately as possible • Hybrid – Abstract some of the biochemical details away while modeling others in detail

  31. Example: The Circadian Clock • Many organisms (including humans), employ different “programs” in light and dark conditions • The switch between the two programs is the result of the entrainment of an internal oscillator by external conditions (light and dark) • The internal oscillator is termed the circadian clock

  32. The Circadian clock • Circadian clock can be found in many organisms including cyanobacteria, fungi, flies, mouse and humans • They may have evolved more than once in evolution • While they differ in the biochemical detail, it appears there is an underlying “design principle” in their regulatory logic • This logic allows faithful intrinsic oscillation of these molecular clocks

  33. The Circadian Clock Flies Mammals Fungi In all cases, there are two integrated regulatory loops: A positive and a negative one

  34. A R degradation A R degradation translation UTRA UTRR translation A_RNA R_RNA transcription transcription PA PR A_GENE R_GENE The Circadian Model (Barkai and Leibler, 2000)

  35. The Circadian Model: Dependency on Differential Rates • Oscillations will be achieved only if there is a certain “imbalance” in the rates in the system • “A” related processes are fast (basal and activated transcription, translation, and RNA and protein degradation) • “R” related processes are significantly slower (e.g. almost no basal transcription) • A-R binding is extremely fast • A hybrid approach: A “logical” model is not enough, but a full biochemical one is currently impossible

  36. The Circadian Clock: System -language(psifcp).-include(rates).global(pA(R1),bA(R2),transcribeA(R13),utrA(R3), degmA(R4),degpA(R12),pR(R5),rbs(R6),bR(R8), transcribeR(R14),utrR(R9),degmR(R10),degpR(R11)).System::= activator#A_GENE | repressor#R_GENE | timer#Timer(transcribeA) | timer#Timer(transcribeR) | machineries#BASAL_TRANSCRIPTION | machineries#BASAL_TRANSLATION | machineries#RNA_DEGRADATION | machineries#PROTEIN_DEGRADATION . clock.cp

  37. The Circadian Clock: “Gene A”, “RNA A” -language(psifcp).-include(rates).global(pA(R1),bA(R2),transcribeA(R13),utrA(R3),degmA(R4),degpA(R12),pR(R5),rbs(R6)).A_GENE::= << PROMOTED_A + BASAL_A . PROMOTED_A::= pA ? {unbind_A} , ACTIVATED_TRANSCRIPTION_A(unbind_A) . BASAL_A::= bA ? [] , A_GENE | A_RNA . ACTIVATED_TRANSCRIPTION_A(unbind)::= transcribeA ? [] , A_RNA | ACTIVATED_TRANSCRIPTION_A ; unbind ? [] , A_GENE >> . A_RNA::= << TRANSLATION_A + DEGRADATION_mA . TRANSLATION_A::= utrA ? [] , A_RNA | A_PROTEIN . DEGRADATION_mA::= degmA ? [] , true >> . activator.cp

  38. Syntax Note: Summing Processes • Processes with a guarded choice construct are called normal processes • Such processes may be summed together, to create a third process, with a combined choice construct, e.g. B::= z ? [] , B1 ; w ? [] , B2 . A::= x ? [] , A1 ; y ? [] , A2 . C::= A + B .x ? [] , A1 ; y ? [] , A2 ;z ? [] , B1 ; w ? [] , B2 .

  39. The Circadian Clock: “Protein A” A_PROTEIN::= << release_AR(infinite),unbind_A_pA(R7a),unbind_A_pR(R7b). PROMOTION_AR + BINDING_R + DEGRADATION_A .PROMOTION_AR::= pA ! {unbind_A_pA} , unbind_A_pA ! [] , A_PROTEIN ; pR ! {unbind_A_pR} , unbind_A_pR ! [] , A_PROTEIN .BINDING_R::= rbs ! {release_AR} , BOUND_A_PROTEIN .BOUND_A_PROTEIN::= degpA ? [] , release_AR ! [] , true ; release_AR ? [] , A_PROTEIN .DEGRADATION_A::= degpA ? [] , true >> . activator.cp

  40. The Circadian Clock: “Gene R”, “RNA R” -language(psifcp).-include(rates).global(bR(R8),transcribeR(R14),utrR(R9),degmR(R10),degpR(R11),pR(R5),rbs(R6)).R_GENE::= << PROMOTED_R + BASAL_R . PROMOTED_R::= pR ? {unbind_A} , ACTIVATED_TRANSCRIPTION_R(unbind_A) . BASAL_R::= bR ? [] , R_GENE | R_RNA . ACTIVATED_TRANSCRIPTION_R(unbind)::= transcribeR ? [] , R_RNA | ACTIVATED_TRANSCRIPTION_R ; unbind ? [] , R_GENE >> .R_RNA::= << TRANSLATION_R + DEGRADATION_mR .TRANSLATION_R::= utrR ? [] , R_RNA | R_PROTEIN .DEGRADATION_mR::= degmR ? [] , true >> . repressor.cp

  41. The Circadian Clock: “Protein R” R_PROTEIN::=<< BINDING_A + DEGRADATION_R . BINDING_A::= rbs ? {release_A} , BOUND_R_PROTEIN(release_A) . BOUND_R_PROTEIN(release)::= release ? [] , R_PROTEIN ; degpR ? [] , release ! [] , true . DEGRADATION_R::= degpR ? [] , true >> . repressor.cp

  42. The Circadian Clock: Machineries -language(psifcp).-include(rates).global(bA(R2),bR(R8),utrA(R3),utrR(R9),degmA(R4),degmR(R10), degpA(R12), degpR(R11)).BASAL_TRANSCRIPTION::= bA ! [] , BASAL_TRANSCRIPTION ; bR ! [] , BASAL_TRANSCRIPTION .BASAL_TRANSLATION::= utrA ! [] , BASAL_TRANSLATION ; utrR ! [] , BASAL_TRANSLATION .RNA_DEGRADATION::= degmA ! [] , RNA_DEGRADATION ; degmR ! [] , RNA_DEGRADATION .PROTEIN_DEGRADATION::= degpA ! [] , PROTEIN_DEGRADATION ; degpR ! [] , PROTEIN_DEGRADATION . machineries.cp

  43. The Circadian Clock: Rates rates.cp * private

  44. Basal Transcription A_GENE | R_GENE | Timer(transcribeA) | Timer(transcribeR) | BASAL_TRANSCRIPTION | BASAL_TRANSLATION |RNA_DEGRADATION | PROTEIN_DEGRADATION pA ? {unbind_A} , ACTIVATED_TRANSCRIPTION_A(unbind_A) ;bA ? [] , A_GENE | A_RNA | pR ? {unbind_A} , ACTIVATED_TRANSCRIPTION_R(unbind_A) ;bR ? [] , R_GENE | R_RNA |bA ! [] , BASAL_TRANSCRIPTION ; bR ! [] , BASAL_TRANSCRIPTION bA (4) bR (0.001) A_GENE | A_RNA | R_GENE | BASAL_TRANSCRIPTION | … A_GENE | R_GENE | R_RNA | BASAL_TRANSCRIPTION | …

  45. Translation and RNA degradation … | A_RNA | R_RNA | … | BASAL_TRANSLATION | RNA_DEGRADATION utrA ? [] , A_RNA | A_PROTEIN ; degmA ? [] , true |utrR ? [] , R_RNA | R_PROTEIN ; degmR ? [] , true |utrA ! [] , BASAL_TRANSLATION ;utrR ! [] , BASAL_TRANSLATION |degmA ! [] , RNA_DEGRADATION ; degmR ! [] , RNA_DEGRADATION degmR (0.02) utrA (1) degmA (1) utrR (0.1) A_RNA | A_PROTEIN | R_RNA | BASAL_TRANSLATION | RNA_DEGRADATION . R_RNA | BASAL_TRANSLATION | RNA_DEGRADATION . A_RNA | R_RNA | A_PROTEIN | BASAL_TRANSLATION | RNA_DEGRADATION . A_RNA | BASAL_TRANSLATION | RNA_DEGRADATION .

  46. pA ! {unbind_A_pA} , unbind_A_pA ! [] , A_PROTEIN ;pR ! {unbind_A_pR} , unbind_A_pR ! [] , A_PROTEIN ;rbs ! {release_AR} , BOUND_A_PROTEIN ;degpA ? [] , true |rbs ? {release_A} , BOUND_R_PROTEIN(release_A) ;degpR ? [] , true |pA ? {unbind_A} , ACTIVATED_TRANSCRIPTION_A(unbind_A) ;bA ? [] , A_GENE | A_RNA |pR ? {unbind_A} , ACTIVATED_TRANSCRIPTION_R(unbind_A) ;bR ? [] , R_GENE | R_RNA |degpA ! [] , PROTEIN_DEGRADATION ;degpR ! [] , PROTEIN_DEGRADATION A R GA GR PDEG Protein A and R A_PROTEIN | R_PROTEIN | A_GENE | R_GENE | PROTEIN_DEGRADATION R - deg machin. A - deg machin. A - R A - GA A - GR pA(10) pR(10) rbs(100) degpA(0.1) degpR(0.01)

  47. Activated Transcription A_PROTEIN | A_GENE | R_GENE pA ! {unbind_A_pA} , unbind_A_pA ! [] , A_PROTEIN ;pR ! {unbind_A_pR} , unbind_A_pR ! [] , A_PROTEIN ;rbs ! {release_AR} , BOUND_A_PROTEIN ;degpA ? [] , true |pA ? {unbind_A} , ACTIVATED_TRANSCRIPTION_A(unbind_A) ;bA ? [] , A_GENE | A_RNA |pR ? {unbind_A} , ACTIVATED_TRANSCRIPTION_R(unbind_A) ;bR ? [] , R_GENE | R_RNA pA(10) pR(10) unbind_A_pA ! [] , A_PROTEIN |ACTIVATED_TRANSCRIPTION_A(unbind_A_pA) |R_GENE unbind_A_pR ! [] , A_PROTEIN |ACTIVATED_TRANSCRIPTION_R(unbind_A_pR) |A_GENE

  48. Activated Transcription pA(10) unbind_A_pA ! [] , A_PROTEIN |ACTIVATED_TRANSCRIPTION_A(unbind_A_pA) |Timer(transcribeA) unbind_A_pA ! [] , A_PROTEIN |transcribeA ? [] , A_RNA | ACTIVATED_TRANSCRIPTION_A ;unbind_A_pA? [] , A_GENE |transcribeA ! [] , Timer Transcription event. RNA molecule released. Protein_A remains bound to promoter A Unbinding event. Protein_A released from promoter A transcribeA(40) unbind_A_pA(10) unbind_A_pA ! [] , A_PROTEIN |A_RNA | ACTIVATED_TRANSCRIPTION_A |Timer(transcribeA) A_PROTEIN |A_GENE | Timer(transcribeA)

  49. A_PROTEIN | R_PROTEIN | … A-R Binding pA ! {unbind_A_pA} , unbind_A_pA ! [] , A_PROTEIN ;pR ! {unbind_A_pR} , unbind_A_pR ! [] , A_PROTEIN ;rbs ! {release_AR} , BOUND_A_PROTEIN ;degpA ? [] , true |rbs ? {release_A} , BOUND_R_PROTEIN(release_A) ;degpR ? [] , true | … rbs(100) BOUND_A_PROTEIN | BOUND_R_PROTEIN(release_AR) | PROTEIN_DEGRADATION degpA ? [] , release_AR ! [] ,true ; release_AR ? [] , A_PROTEIN |release_AR ? [] , R_PROTEIN ; degpR ? [] , release_AR ! [] , true |degpA ! [] , PROTEIN_DEGRADATION ;degpR ! [] , PROTEIN_DEGRADATION degpR(0.01)followed byrelease_AR degpA(0.1) followed byrelease_AR R_PROTEIN | PROTEIN_DEGRADATION A_PROTEIN | PROTEIN_DEGRADATION

  50. A R degradation A R degradation translation UTRA UTRR translation A_RNA R_RNA transcription transcription PA PR A_GENE R_GENE The Circadian Model (Barkai and Leibler, 2000)

More Related